Neurological disorders such as stroke, multiple sclerosis (MS), Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Fibromyalgia (Smith et al., Ann Pharmacother 35(6):702-706, 2001), Parkinson's disease, and Huntington's disease (Kim et al., Chapter 1 in CNS Neuroprotection. Springer, N.Y. pp. 3-36, 2002), eye pathologies, and traumatic brain injuries affect a large portion of the population, but efficient pharmacological treatments are still lacking. One crucial mechanism underlying these diseases is excitotoxicity conveyed by NMDA-type of glutamate receptors (NMDAR). Recent research has shown that this mechanism offers great potential for the development of new pharmacological treatments.
The term excitotoxicity refers to the pathological process by which neurons are damaged and killed by the overactivation of receptors for the excitatory neurotransmitter glutamate, such as the NMDA receptor and AMPA receptor. Excitotoxins like NMDA and kainic acid (KA) which bind to these receptors, as well as pathologically high levels of glutamate, can cause excitotoxicity by allowing high levels of calcium ions to enter the cell (Manev et al., Mol Pharmacol 36(1):106-112, 1989). Ca++ influx into cells activates a number of enzymes, including phospholipases, endonucleases, and proteases such as calpain. Activation of these enzymes leads to irreversible damage to various cell structures such as components of the cytoskeleton, membranes, and DNA.
The neurotoxic effects of glutamate were first observed in 1954 by T. Hayashi, a Japanese scientist who noted that direct application of glutamate to the CNS cause d seizure activity, though this report went unnoticed for several years. The toxicity of glutamate was then observed by D. R. Lucas and J. P. Newhouse in 1957 when the feeding of monosodium glutamate to newborn mice destroyed the neurons in the inner layers of the retina (Lucas et al., AMA Arch Ophthalmol 58(2):193-201, 1957). Later, in 1969, John Olney discovered the phenomenon wasn't restricted to the retina but occurred throughout the brain and coined the term excitotoxicity. He also assessed that cell death was restricted to postsynaptic neurons, that glutamate agonists were as neurotoxic as their efficiency to activate glutamate receptors, and that glutamate antagonists could stop the neurotoxicity (Olney, Science 164(880):719-721, 1969).
The major excitotoxin in the brain, glutamate, is paradoxically also the major excitatory neurotransmitter in the mammalian CNS (Temple et al., Chapter 4 in Head Trauma: Basic, Preclinical, and Clinical Directions. John Wiley and Sons, Inc., New York. pp. 87-113, 2001). During normal synaptic transmission, glutamate concentration can be increased up to 1 mM in the synaptic cleft, which is rapidly decreased in the lapse of milliseconds. When the glutamate concentration around the synaptic cleft cannot be decreased or reaches higher levels, the overexcited neuron kills itself by a process called apoptosis; alternatively a necrosis cell death can also occur. This pathologic phenomenon is also frequently found after brain injury.
In brain trauma, or stroke, ischemia often results, reducing blood flow to inadequate levels. Ischemia is then followed by accumulation of glutamate and aspartate in the extracellular fluid, causing cell death, which is aggravated by the lack of oxygen and glucose. The biochemical cascade resulting from ischemia and involving excitotoxicity is called the ischemic cascade. Once the ischemic cascade triggers excitotoxicity, an influx of Ca++ ensues to activate a number of cell damaging enzymes.
Another damaging result of excess calcium in the cytosol is the opening of the mitochondrial permeability transition pore, a pore in the membrane of mitochondria that opens when the organelles absorb too much calcium. Opening of the pore causes mitochondria to swell and release proteins that can lead to apoptosis. The pore can also cause mitochondria to release more calcium. In addition, production of adenosine triphosphate (ATP) may be stopped, and ATP synthase may in fact begin hydrolysing ATP instead of producing it (Stavrovskaya et al., Free Radical Biology and Medicine. Volume 38, Issue 6, pages 687-697).
Inadequate ATP production resulting from brain trauma can eliminate electrochemical gradients of certain ions. Glutamate transporters require the maintenance of these ion gradients in order to remove glutamate from the extracellular space. The loss of ion gradients results not only in the halting of glutamate uptake, but also in the reversal of the transporters, causing them to release glutamate and aspartate into the extracellular space. This results in a buildup of glutamate and further damaging activation of glutamate receptors (Siegel et al., Basic Neurochemistry: Molecular, Cellular, and Medical Aspects, 6th ed., Lippincott, Williams & Wilkins, Philadelphia, 1999).
At the molecular level, calcium influx is not the only event responsible for apoptosis induced by excitoxicity. Recently it has been noted that extrasynaptic NMDA receptor activation, triggered by glutamate exposure or hypoxic/ischemic conditions, activates a CREB (cAMP response element binding protein) shut-off, which in turn, causes loss of mitochondrial membrane potential and apoptosis (Hardingham et al., Nat Neurosci 5(5):405-414, 2002). On the other hand, activation of synaptic NMDA receptors only activates the CREB pathway which activates BDNF (brain-derived neurotrophic factor), not apoptosis.
Glutamate antagonists have been known to stop neurotoxicity due to excitotoxins. Methods for treating neurological injuries and neurodegenerative diseases have largely focused on finding more potent glutamate antagonists. However, this approach has not been very effective in clinical settings.
For example, the blood-clot dissolver tissue plasminogen activator (TPA) can reduce the disability of people who survive an ischemic stroke and has been an important breakthrough in the treatment of acute neurological disorders. However, there is only a 3-hour window within which a patient must receive the TPA treatment from the time of the ischemic insult in order for the treatment to be effective. Today, more than 40% of stroke patients do not reach medical personnel within this critical time period. Furthermore, patients with hemorrhage must be excluded from TPA treatments. In view of these statistics, methods for widening this critical treatment window for neuroprotective treatments have been an area of intensive research. And because neural damage results not only from hypoxic cell death, but also from excitotoxic cell death, these methods have aimed at inhibiting glutamate release or calpain activity.
Recently, calpain inhibitors have been found to be neuroprotective in animal models of stroke (Bartus et al., Stroke 25(11):2265-2270, 1994; Goll et al., Physiol Rev 83(3):731-801, 2003; Liebetrau et al., Neurol Res 27(5):466-470, 2005; Markgraf et al., Stroke 29(1):152-158, 1998; Wu and Lynch, Mol Neurobiol 33(3):215-236, 2006); however, no calpain inhibitor has reached the clinic. This is likely due to the relatively low specificity of existing calpain inhibitors as well as to the broad spectrum of calpain substrates and functions in which this family of enzymes is implicated. Thus, there still exits a need for more efficient pharmacological treatments that have reduced side effects.